Z-138: a new mature B-cell acute lymphoblastic leukemia cell line from a patient with transformed chronic lymphocytic leukemia

Leukemia Research

PERGAMON

Leukemia Research 22 (1998) 341-353

Z-l 38: a new mature B-cell acute lymphoblastic leukemia cell line from a patient with transformed chronic lymphocytic leukemia Z. Estrov a,*, M. Talpaz a, S. Ku a, D. Harris a, Q. Van a, M. Beran b, C. Hirsch-Ginsberg c, Y. Huh c, G. Yee c, R. Kurzrock a a Department of Bioimmunotherapy,

The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, P.O. Box 302, Houston, TX 77030, USA b Department of Hematology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA ‘Division of Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA

Received 1 October 1997; accepted 31 October 1997

Abstract We describe a new mature B-cell acute lymphoblastic leukemia (ALL) cell line designated Z-138 that was derived from a patient with chronic lymphocytic leukemia (CLL) whose disease underwent transformation to a rare, aggressive form of mature B-cell ALL. This cell line has an L3 morphology, ultrastructural characteristics of lymphoblasts, B-lineage surface markers and an immunoglobulin heavy-chain gene rearrangement identical to the rearrangement observed in the patient’s blasts from whom the cell line was derived. Z-138 cells produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and high levels of granulocyte-CSF (G-CSF), but they do not exhibit a proliferative response to either cytokine. Both the patient’s lymphoblasts and Z-138 cells exhibited cytogenetic abnormalities including t(8;14), t(14;lS) and a chromosome 11 abnormality similar to the t(l1;14) of the parental cells, resulting in marked overexpression of cyclin Dl (BCL-1 (PRADl)) mRNA in Z-138 cells. Since these karyotypic anomalies have been associated with low grade (t(14;18)), intermediate grade (t(l1;14)) and high grade (t&14)) lymphomas, their development may be involved in the unusual aggressive transformation of this patient’s CLL. 0 1998 Elsevier Science Ltd. All rights reserved. Keywords: Cell line; Chronic lymphocytic leukemia; Acute lymphoblastic leukemia

1. Introduction Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion and accumulation of leukemic cells

Abbreviations: ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; GM-CSF, granulocyte-macrophage colonystimulating factor; G-CSF, granulocyte colony-stimulating factor; PRADl, cyclin Dl/BCL-1; EBV, Epstein-Barr virus; BM, bone marrow; Hb, Hemoglobin; WBC, white blood cell count; TdT, terminal deoxynucleotidyl transferase; MPO, myeloperoxidase; NASD, chloroacetate; NSE, cc-naphthyl butyrate esterase;PAS, periodic acidSchiff; EM, electron microscope; N/C, nucleus to cytoplasm; J,, heavy-chain gene; CT, computerized tomography; FCS, fetal calf serum; pH, phosphate buffer; MHC, major-histocompatibility-complex; Ph’ Philadelphia chromosome; MTC, major translocation cluster; TGF-l3, transforming growth factor-/?; sIg, surface membrane I. *Corresponding author. Tel.: + 1 713 7941675; fax: + 1 713 7962173.

0145-2126/98/$19.000 1998 Elsevier Science Ltd. All rights reserved. PII: SOl45-2126(97)00191-4

with B-lymphocyte characteristics. Approximately 10% of all patients with B-CLL develop more aggressive B-cell neoplasms including diffuse large cell lymphoma (Richter’s syndrome), prolymphocytic leukemia, multiple myeloma and acute lymphoblastic leukemia (ALL) [l]. The occurrence of ALL in patients with CLL is rare and limited to case reports [2-51. The mechanisms underlying the transformation of CLL are largely unknown. A rare form of mature B-cell ALL has been identified in recent years [6]. This subset of ALL is a distinct clinical entity with an aggressive course and a poor outcome. The lymphoblasts in mature B-cell ALL present surface immunoglobulin

(Ig) with a M chain

and cytogenetic abnormalities of chromosome 8 including t&14). To our knowledge transformation of CLL to this rare form

previously.

of ALL

has not been described

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In this report, we describe a newly established Epstein-Barr virus (EBV)-negative mature B-cell ALL line designated Z-138. This line, derived from a patient in whom mature B-cell ALL evolved from CLL, exhibits an L3 morphology, ultrastructural features of a B lymphoblast, Ig heavy-chain gene rearrangement and B-lineage surface markers. Z-138 also exhibits several cytogenetic aberrations, including chromosome 8 abnormalities (t(8;14)), t(14;18) and chromosome 11 abnormalities (t(l1;14)), that are identical to those of the parent lymphoblasts from which the cell line was derived. Z-138 cells also produce granulocytemacrophage colony-stimulating factor (GM-CSF) and large quantities of granulocyte colony-stimulating factor (G-CSF) but do not proliferate in response to these cytokines. 2. Materials and methods 2.1. Source of the cell line The cell line Z-138 was derived from bone marrow (BM) cells of a 70-year-old Caucasian man who was diagnosed with CLL in December 1987. At that time, his hemoglobin (Hb) level was 11.5 gm dl - ‘; his platelet count was 129 x lo9 1- ’ and his white blood cell count (WBC) was 83 x lo9 1- ’ with 90% small lymphocytes. His BM exhibited a 60% cellularity. All hematopoietic lineages were present and 50% of the cells were small lymphocytes, consistent with CLL. The patient was first treated with chlorambucil and prednisone with a poor response and at a later stage with a combination of cyclophosphamide, vincristine and prednisone. In December 1988 the patient underwent a splenectomy because he had massive splenic enlargement and was experiencing significant discomfort. In May 1990, the patient was referred to our institution because of an increase in his WBC and a lack of response to chemotherapy. Upon admission, the patient complained of extreme weakness. His physical examination revealed cervical, axillary and inguinal lymphadenopathy, rales were heard during auscultation of both lungs and pretibial pitting edema was found in the lower extremities. The patient’s Hb level at this time was 9.9 gm dl- l his platelet count was 82 x lo9 1- ’ and his WBC was 900 x lo9 1- I. Most of the phipheral blood cells examined were small lymphocytes and 68% of them had a lymphoblastic appearance. A BM aspirate exhibited 80% cellularity with 82% of the cells having an L2 lymphoblastic morphology. Most of the remaining cells were mature lymphocytes, as consistent with a leukemic transformation of CLL. The blasts did not stain with terminal deoxynucleotidyl transferase (TdT), myeloperoxidase (MPO), chloroacetate (NASD), a-naphthyl bu-

Research 22 (1998) 341-353

tyrate esterase (NSE) or periodic acid-Schiff (PAS). A BM biopsy indicated 80% cellularity with = 80% of the cells being lymphoblastic with irregular nuclear profiles. Electron microscope (EM) examination revealed a large population of small blasts with round to slightly indented nuclei containing course heterochromatin and small prominent nucleoli. The nucleus to cytoplasm (N/C) ratio was medium with a few cytoplasmic organelles and degenerating mitochondria. Numerous mature lymphocytes were also seen. Ultrastructural staining was negative for MPO. Immunophenotyping studies showed small and large lymphocytes expressing CD19 (98%), CD20 (98%) CD5 (86%) HLA-DR (98%) and IgG M (97%) but not myeloid or T-cell markers. Cytogenetic studies detected two hyperdiploid clones: [48, XY, + 3, + 13, 8q - , t(llq - ;14q +), t(14q + ;18q - )] in 17 metaphases and [49, XY, + 3, +12, +13, 8q-, t(llq-;14q+), t(14q+;18q-)] in five metaphases. Molecular analysis identified a rearrangement of the immunoglobuiin heavy-chain gene (Jn), consistent with a monoclonal population of B-lineage cells. No rearrangement was found with a probe for the c-myc oncogene. The patient underwent repeated apheresis and then received treatment with a combination of doxorubicin, vincristine and dexamethasone. In August 1990, the patient was admitted because of confusion, back pain and fever. A lumbar puncture revealed 2700 lymphoblasts cm - 3 and a computerized tomography (CT) scan demonstrated leptomeningeal disease with parenchyma1 involvement. An Ommaya reservoir was implanted and cytosine arabinoside and hydrocortisone were administered intrathecally. In addition, the patient’s systemic treatment was changed to a combination of cyclophosphamide, doxorubicin, vincristine and prednisone. A significant improvement was achieved and the patient was discharged from the hospital. In September 1990, the patient experienced ataxia and short episodes of weakness in his left arm. Leukemic cells were detected again in his cerebrospinal fluid and the intrathecal therapy was changed to a combination of methotrexate and hydrocortisone. Fludarabine and prednisolone were administered systemically. In November 1990, the patient died of septicemia despite intravenous antibiotic therapy. 2.2. Cell culture A BM aspirate was obtained from the patient after the patient had given informed consent in accordance with the procedures of The University of Texas, MD Anderson Cancer Center’s Institutional Review Board. Heparinized BM cells were layered over Ficoll-Hypaque (Pharmacia, Piscataway, NY) and centrifuged (400 x g, 4°C) for 20 min to remove neutrophils and red blood cells. Low-density BM cells (1 x lo6 ml- ‘)

2. Estrov et al. /Leukemia

were cultured in RPM1 1640 (Gibco, Grand Island, NY) and supplemented with 10% fetal calf serum (FCS; Flow Laboratories, McLean, VA). Cultures were maintained in 25-c& tissue culture flasks (Becton Dickinson, Oxnard, CA) and were fed by replacing 80% of the medium with an equal volume of fresh complete medium every 3 days for several months. Cultures were maintained at 37°C with 5% CO, in air within a humidified atmosphere. Cells from the established line were maintained in logarithmic growth prior to testing. 2.3. Light microscopy

Z-138 cells were cytocentrifuged on glass slides, air dried and stained with Wright Giemsa and with MPO, NASD, NSE and PAS by standard techniques. TdT activity was assessedby indirect immunofluorescence. 2.4. Electron microscopy (EM)

The cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h at 25°C. For ultrastructural identification of MPO, the cells were incubated in 33’-diaminobenzidine, according to the method described by Graham and Karnovsky [7]. After incubation, cells were postfixed in 2% osmium tetroxide in phosphate buffer for 30 min at 4°C then subsequently dehydrated with a graded acetone series and embedded with EPON. The sections were cut with a Reichert ultracut E (Reichert Optische Werke AG, Wien, Austria) and counterstained with uranyl acetate and lead citrate. Sections were viewed on a Jeol 1200 electron microscope (Jeol, Tokyo, Japan) at 40 kev. Sections used for the identification of MPO were not counterstained to facilitate the visualization of the reaction product. The percent of peroxidase-positive blasts was derived by counting 200 blasts on a single section.

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using a previously described method [lo]. Briefly, cells at the logarithmic phase of growth were incubated for 25 min in demecolcine (Colcemid; final concentration, 0.06 ug ml - ‘), rinsed twice in Hank’s balanced salt solution and exposed to a hypotonic solution (0.075 mol I- ’ Kcl) at room temperature for a total of 32 min, which included periods of mixing, standing and centrifugation. The cells were then fixed in methanolacetic acid (3:1, vol/vol) for 15 min. Slides were prepared using a flaming technique and were allowed to age for O-7 days. Metaphase preparations were Gbanded by a modification of the trypsin method of Seabright [lo] and chromosomes were identified and assigned according to the International System of Human Cytogenetic Nomenclature [ 111. 2.7. Control cell lines

Control cell lines included SW954 (a vulvar carcinoma cell line previously shown to have high levels of PRADl RNA [12]) and SW765 (a cervical carcinoma cell line (ATCC, Rockville, MD) as well as ALL-l cells (a Philadelphia chromosome (Ph’)-positive ALL cell line (provided by Dr G. Rovera, Wistar Institute, Philadelphia, PA), RCK8, a (diffuse histiocytic lymphoma cell line-provided by Dr I. Kubonishi, Kochi, Japan)), K562 cells (a Ph’-positive CML blast crisis cell line) and HL-60 (a Ph’-negative leukemia cell line (ATCC)). 2.8. Immune complex kinase assay

Immunophenotyping was carried out with flow cytometry using antibodies against the following: T-cell antigens (CD2, CD3, CD4, CD5, CD7 and CD8), B-cell antigens (CDlO, CD19 and CD20), an early progenitor marker (CD34), the major-histocompatibility-complex (MHC)-related antigen-HLA-DR and myeloid markers (CD13, CD14, CD15, CDllb and CD33). Expression of a specific marker was considered positive if it was expressed on 2 20% of the leukemic cells [8,9].

Occasionally patients with a q34 abornamality demonstrate formation of bcr-abl, despite a lack of cytogenetic evidence of a chromosome 22 abnormality. Therefore, we performed experiments to detect bcr-abl in our cell line. p190bcr-ab’and p210bc’“b’ can be detected in vitro by exploiting their tyrosine phosphokinase enzymatic activity in the immune complex kinase assay. The antiserum used for this assay was antiab1389-403, which is a rabbit polyclonal serum made against the predicted hydrophilic domain of v-abl [13]. The immune complex kinase assay was performed on 5 x lo6 cells. K562 cells served as a positive control and HL-60 cells were used as a negative control. To ensure that the 210-kD and 190-kD bands on the gel represented a protein recognized by the anti-abl serum (rather than background phosphorylation), alternate cell samples were incubated with anti-abl serum and blocking cognate peptide. The method for the immune complex kinase assay is identical to the one previously published in detail by our laboratory [ 13- 151.

2.6. Cytogenetic analysis

2.9. Probes

2.5. Surface marker analysis

Cytogenetic analysis of the cell line was performed

The J, probe was a 2.9-kb HindIII-EcoRI

insert

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(provided by Dr A. Deisseroth, M.D., Anderson Cancer Center, Houston, TX). The EBV probe was the B95-8 BumHI K fragment containing the coding region for nuclear antigen-l (provided by Dr J. Hearing) [16]. The PRADl probe (pPI-8) was a 1.4-kb cDNA probe that was donated by Dr A. Arnold and represents the API-4 insert [17]. /3-actin is a 1.l-kb cDNA probe (HHC189) (ATCC) that was used as a control for Northern blot analysis of RNA. Five different probes were used to search for rearrangement in BCL-1 or PRADl: &l-lb, which is a 2.1-kb Sac1 fragment of the cDNA from the major translocation cluster (MTC) region from bcl-1 (provided by Dr Y. Tsujimoto) [18] bcl-la, a 0.8-kb fragment upstream from &l-lb; and bcl-l/pG14 (provided by Dr Tsujimoto) which corresponds to a sequence 28 kb upstream from &Z-lb [ 191; PRADl (pPI-B), a 1.4-kb EcoRI fragment (MPI-4) of the partial cDNA clone of the PRADl gene [17] and PRADl pDy-12, a 500bp insert of genomic DNA located about 15 kb upstream of the PRADl exon 1 (both provided by Dr A. Arnold). 2.10. Southern blotting for DNA analysis High molecular DNA was prepared as previously described [20]. Fifteen micrograms of DNA was digested with restriction endonucleases in conditions recommended by the supplier (International Biotechnologies, New Haven, CT) electrophoresed on 0.8% agarose gel, blotted and hybridized according to the methods of Southern [20]. The probes were labeled by oligo primer extention to a specific activity of l-3 x lo9 cpm ug-’ of DNA [21]. After hybridization, filters were washed at 60°C for 1 h with a solution of 0.1 x SSC (1 x SSC = 0.15 mol 1- ’ sodium chloride plus 0.015 mol l- ’ sodium citrate) and 0.1% sodium dodecyl sulfate. Filters were then dried and autoradiographed. The intensity of the signal was compared by densitometry measurements using a Beckman Du-70 spectrophotometer. 2.11. Northern blotting for RNA analysis Poly(A) + -selected mRNA (4 ug per lane) was sizefractionated by electrophoresis in 1.l% agarose gels containing 2.2 mol 1- ’ formaldehyde and transferred to nitrocellulose [22]. The filters were hybridized with 32P-radiolabeled DNA probes, washed and radiographed in the same conditions as were the DNA filters. Preparation of probes and hybridization conditions have been described previously [21]. The intensity of the signal was compared by densitometry measurements using a Beckman Du-70 spectrophotometer and normalization for the signal for the /3-actin control probe.

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2.12. Polymerase chain reaction (PCR) PCR was used to identify if a bcr-abl transcript was produced by Z-138 cells. Total cellular RNA was extracted using previously described methods [23]. RNA from the Ph’-positive CML blast crisis cell line K562 was used as control. The ALL-l cell line was used as a positive control for the transcript encoding ~190~“~ abl (e,-a2 junction). HL-60 and normal human endometrial RNA were used as negative controls. One microgram of total RNA from each sample was used for amplification reactions. The amplification method and primers have been previously described [16,23]. Amplification was carried out for 40 cycles. Contamination has proven to be a significant problem in some laboratories using this exquisitely sensitive PCR technique, consequently, we took the following precautions to ensure the accuracy of our results: (i) the thermal cycler was kept in a separate laboratory, away from the room where cell collection, RNA processing and cDNA synthesis were carried out; (ii) no amplified samples were allowed to be brought back into the room where RNA processing was carried out; (iii) at least one negative control was run for each experiment; and (iv) each sample was run on at least two different occasions. 2.13. Enzyme-linked immunosorbent assay (ELISA) We measured the concentrations of IL-lp, IL-6, tumor necrosis factor-a (TNF-a), transforming growth factor-/I (TGF-P), G-CSF and GM-CSF in the supernatants and lysates of Z-138 cells using ELISA kits. The ELISAs were performed using specific kits for IL-P, IL-6, TNF-aa, TGF-P, GM- CSF (Cistron Biotechnology, Pine Brook, NJ) and G-CSF (R&D Systems, Minneapolis, MN) as previously described [24]. Briefly, supernatants and 2 x lo7 cells were frozen at - 20°C in duplicate then tested later for cytokine content. Cell lysates, supernatants and standard dilutions of each cytokine were added to test wells in duplicate then incubated with rabbit antiserum for 2 h. After they were washed, the wells were incubated for 30 min with goat anti-rabbit IgG conjugated with a horseradish peroxidase enzyme. A substrate (ophenylenediamine dissolved in a 3% hydrogen peroxide solution) was added to the test wells following vigorous washing; 4 N sulfuric acid was then added. Within 15 min of adding the sulfuric acid, the color intensity was read at a wavelength of 490 nm using a microplate autoreader (model EL-309; Bio-Tek, Winooski, VT). The average net optical density of the standard cytokine concentration was plotted and the cytokine amount in each sample was determined by interpolation from the standard curve.

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Fig. 1. Morphological characteristics of Z-138 cells. Wright-Giemsa staining: Z-138 cells show a moderate amount of basophilic cytoplasm with numerous cytoplasmic vacuoles. The nuclei contain one or more prominent nucleoli.

2.14. Cell line clonogenic assay

Z-138 cells at their logarithmic phase were cultured in quadruplicate in 35-mm petri dishes (Nunc, Naperville, IL) at a concentration of 2 x lo4 cells ml - ’ in 0.8% (v/v) methylcellulose in RPM1 (GIBCO) with 10% FCS and incubated at 37°C in a humidified atmosphere of 5% CO, with air. Cultures were evaluated on day 6 using an inverted microscope [25]. A cluster of more than 40 cells was defined as a colony. In various experiments the following cytokines and their corresponding neutralizing antibodies were added to the cultures: 25-125 ng ml-’ of the recombinant human (rh)GM-CSF (Immunex, Seattle, WA), 50-100 ng ml1 of rhG-CSF (Amgen, Thousand Oaks, CA), 0.30-3.0 ng ml-’ of mouse anti-human GM-CSF neutralizing antibodies (Genzyme, Cambridge, MA), 10 ng ml - i of anti-G-CSF antibodies (Amgen) and 100 ng ml-’ of anti-G-CSF antibodies (R&D Systems). These concentrations were chosen based on our previous experience with these agents [26,27]. 3. Results 3.1. Establishment of the Z-138 all cell line

Persistent cell proliferation was observed in one flask of the cell line following 3 months of incubation. The cells were maintained by replacement with fresh medium (RPM1 1640 supplemented with 10% FCS) at 3- to 4-day intervals. At the time of writing, the Z-138

cells were 75 months old. The doubling time of Z-138 cells is 18-24 h. The cells are density-independent and can proliferate in a limiting-dilution assay. Tests of the Z-138 cells were negative for mycoplasma. All cultures showed a homogenous cellular population. There was no evidence of contamination with other cells. 3.2. Cell characterization

studies

3.2.1. Morphology

Z-138 cells had a moderate amount of basophilic cytoplasm with numerous cytoplasmic vacuoles. TdT, MOP and NASD cytochemical stains were negative. Rare coarse granular positive staining was found with PAS. The nuclei had one or more prominent nucleoli (Fig. 1). The overall morphology was reminiscent of ALL FAB L3 [28]. 3.2.2. Electron microscopy

Ultrastructurally, the Z-l 38 cells displayed the characteristic morphology of a lymphoblast. The cells appeared homogenous in size (8 uM in diameter) and their nuclear configuration was irregular. The cells’ nuclear chromatin appeared to be coarse and condensed. Nucleoli were present but were not prominent. The moderate cytoplasm contained scant organelles with scattered mitochondria, occasional golgi appartuses and rare endoplasmic reticulum. Prominent lipid vacuoles were present in the cytoplasm of nearly all of the cells as compatible with L3-type lymphoblasts (Fig. 2).

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Fig. 2. Ultrastructural characteristics of Z-138 cells which display prominent lipid vacuoles and nucleus, having an irregular configuration with a coarse and condensed chromatin and occasional nucleoli.

Immunophenotype studies revealed that Z-138 cells expressed B-cell surface markers (CD19, CD20 and IgGM) that were similar to those expressed by the parent BM leukemic blasts (Table 1). In addition, the cell line expressed HLA-DR and CD38 but not the

early hematopoietic precursor marker CD34. Interestingly, CD5 antigen was expressed by the parent blasts but not by Z-138 cells. Neither, Z-138 cells nor their parent blasts expressed,myeloid (CD33, CD1 lb, CD13, CD14) or T-cell surface markers (CD2, CD3, CD4, CD7 and CD8).

Table 1 Percent of cells expressing surface markers

3.2.4. Karyotype analysis

3.2.3. Immwzophenotype analysis

Surface marker

BM blasts

Cell line

HLA-DR CD2 CD3 CD4 CD5 CD7 CD8 CD10 CDllb CD13 CD14 CD15 CD19 CD20 CD22 CD25 CD33 CD34 CD38 CD56 IgM + IgD Ifi K IgG i

98 4.0 2.0 1.0 86 2.0 <1 1.0 2.0 2.0 3.0 <1 98 98 ND 6.0 2.0 <1 ND 1.0 95 1.0 97

99
Comparison of patient’s original marrow blasts with Z-138 cells. BM, bone marrow; ND, not done.

Cytogenetic analysis of the metaphases of fifteen Z-l 38 cells revealed a male chromosome complement with numerical and complex structural abnormalities consistent with ALL. The karyotype was as follows: 48, XY, der(8)t(8;14;?)(q24;q32;?), der(9)t(9;?) (q34;?), del(1 l)(q13q25), + 12, de1(12)(q22q24.1),+ 13, der(l4)t(8;14;?)(q32;q21;?), der(l8)t(l4;18;?)(q32;q21;?) (Fig. 3). 3.2.5. Z-138 cells demonstrate clonal Jh rearrangement

Southern blotting of DNA from Z-138 cells demonstrated clonal J, rearrangement supporting the lymphoid lineage derivation of this leukemia (Fig. 4, lane 2). Comparison of Z-138 cells (Fig. 4, lane 2) with peripheral blood leukocytes from the patient termed D-Z-138 (Fig. 4, lane I), from whom the cell line was derived showed identical J, rearrangements, confirming that the cell line originated from this individual. However, a molecular evolution had occurred in the cell line since the germline bands were no longer seen in the EcoRI-or HindIII-digested DNAs (Fig. 4, panels B and C, lanes 1 and 2) and were very faint in the BamHI-digested DNA (Fig. 4, Panel A, lanes 1 and 2), suggesting a deletion in this region.

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Fig. 3. The karyotype of the parental leukemic cells and the Z-138 cell line. Upper panel: The parental leukemic cells show a hyperdiploid karyotype 48, XY, + 3, + 13, 8q -, t(llq-;14q+), t(l4q +;18q-). Lower panel: The Z-138 cells show a male karyotype with several abnormalities: 48, XY, der(8), t(8;14?)(q24;q32;?),der(9)t(9;?)(q34;?),del(1 l)(q13q25), + 12, del(12)(q22q24.1), + 13, der(14)t(14;18;?)(q32;q21;?), der(l8)t(l4;18;?)(q32;q21;?). The similarities between the two karyotypes (e.g. del(8q), t(9;?), del(l lq), + 13, t(14;18), t(14;?)) illustrate the fact that the Z-138 cell line was derived from the patient’s lymphoblasts.

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A

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B

C

Fig. 4. Southern blot of DNA was hybridized with a JH probe. DNA was extracted from blood derived from patient D-Z-138 (the patient from whom the Z-138 cell line was derived) (lane l), Z-138 cells (lane 2), blood leukocytes taken from a CML patient used as a control (lane 3) and from K562 cells (CML cell line used as a control) (lane 4). Restriction enzymes used included BarnHI (panel A), EcoRI (panel B) and Hind111 (panel C).

3.2.6. Z-138 cells are not EBV transformed

Southern blotting and hybridization with an EBV probe failed to detect EBV sequencesin the Z-138 cells (data not shown), indicating that this was not an EBVtransformed lymphoblastoid line. 3.3. Z-138 cells express high levels of PRADl

mRNA

Z-138 cells were found to express PRADl mRNA at levels over 20 times greater than those in other lymphoid cell lines (Fig. 5). In contrast to squamous cell carcinoma cell lines that frequently expressed high levels of a 4.5-kb PRADl transcript (Fig. 5, lane 1)

Kb

[12], the Z-l 38 cells expressed a 1.7-kb PRADl transcript. Despite the high levels of RNA, we were unable to find bcl-1 or PRADl rearrangement at the DNA level, probably because of the widespread sites of breakpoints for this gene. Southern blotting of DNA and hybridization with a panel of five PRADl/BCLl probes was performed (Figs. 6 and 7). Although an anomalous band was detected in the EcoRI-digested DNA of both Z-l 38 cells and the parent D-Z-138 cells after hybridization with the PRADl pPI-8 probe (Fig. 7, upper panel, lanes 7 and S), only germ line bands were seen in the Ban?HI-(Fig. 7, upper panel, lanes 1 and 2), Ml-(Fig. 4, upper panel, lanes 4 and 5) XbaI-(Fig. 7, lanes 10 and 1l), HindIII-, BglII-, PstIand XhoI-digested DNA lanes (data not shown). These results suggest that the anomalous band represents a polymorphism.

4.5 3.4. Z-138 cells do not express bcr-abl despite a 9q34 anomaly

1.7

2.2

/3-ACTIN

Fig. 5. Northern blot of mRNA hybridized with the PRADl (pPI-8) probe (upper blot) and a control /j’-actin probe (lower blot). Lane 1, SW954 (a vulvar squamous cell carcinoma cell line known to overexpress PRADI); Lane 2, ALL1 cells (Ph’-positive ALL cell line); Lane 3, RCKS (diffuse histiocytic lymphoma cell line; Lane 4, SW756 a cervical carcinoma cell line; and Lane 5, Z-l 38 cells.

The hallmark of the Ph’ translocation (t(9;22)(q34;qll)) is the bcr-abl hybrid gene. Leukemia cells with a cytogenetic anomaly at 9q34 but without a detectable chromosome 22 anomaly can express bcr-abl when analyzed by molecular techniques. Such techniques include the immune complex kinase assay which detects phosphorylated bcr-abl protein and the PCR technique, which detects bcr-abl transcripts by amplification around the b2-a2, b3-a2 and el-a2 cDNA junctions. (These junctions are characteristic of the vast majority of cases with Ph’-positive leukemias [29]. Z138 cells however showed no evidence of an aberrant bcr-abl messageby PCR nor was the p210h”+” or the P190hcr-ab[product detected by the kinase assay (data not shown).

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4. Discussion

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BCLla

BCLlb

BCLI-PG14

Fig. 6. Southern blot of DNA from Z-138 cells (lanes 1, 4 and 7), blood leukocytes from a CML patient used as a control (lanes 2, 5 and 8) and from K562 cells (CML cell line used as a control) (lanes 3, 6 and 9). Restriction enzymes used include BumHI (lanes l-3), EcoRI (lanes 4-6) and Hind111 (lanes 779). Probes for hybridization were BCLla, BCLlb and BCLl-PG14 as shown.

3.5. Z-138 cells produce G-CSF and GM-CSF but not IL-lb, IL-6, TNF-c( and TGF-p

Because myeloid growth factors have been found to be produced and to stimulate ALL cell-line proliferation [30], we used ELISAs to evaluate whether Z-138 cells produce any of these cytokines. We found that Z- 138 cells produce significant amounts of G-CSF and GM-CSF but do not produce IL-lp, IL-6, TNF-CY,or TGF-P (Table 2). 3.6. Z-138 cells do not proliferate G-CSF and GM-CSF

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in response to

We then investigated whether the endogenously produced cytokines stimulate Z-138 cell proliferation. We found, in repeated experiments, that neither G-CSF, GM-CSF, nor their corresponding antibodies affected Z-138 cell growth (data not shown).

The Z-138 cell line has been derived from BM cells obtained from a patient with ALL supervening CLL. The Z-138 cells were similar to the patient’s original BM blasts, having an identical J, rearrangement and similar cytogenetic characteristics. Surface marker analysis and DNA analysis for the presence of EBV confirmed that Z-138 is an EBV-negative ALL B-cell line. Several lines of evidence suggest that the transformation of CLL to ALL represents a clonal evolution of the initial CLL clone. Those include the occurrence of identical Ig isotypes [3], the presence of the same J, rearrangements [1,33,34] and common chromosomal aberrations seen in both tumor populations [34-361. Numerous molecular events may induce leukemic transformation and the development of a rapidly proliferating leukemic clone and an aggressive disease. Both the patient’s blasts and Z-138 cells presented such features. ALL with a mature B-cell phenotype is characterized by the presence of a surface membrane Ig (sIg) and the absence of TdT and FAB-L3 morphology [36]. The lymphoblasts of our patient had an L2 morphology whereas the Z-l 38 cell line had cytoplasmic vacuoles that may have developed in vitro thus showing morphological features reminiscent of ALL FAB L3. Hammami et al. [6] described nine cases of mature B-cell ALL that did not have FAB-L3 morphology. These cases define a distinct clinicopathologic entity with an aggressive clinical course, poor response to treatment and short survival. Similar to our patient, all nine adult patients were men, three of whom had meningeal involvement. Eight evaluated cases had sIg with a Mchain predominance, four cases had cytogenetic abnormalities involving chromosome 8 (including t(8;14)) and one case presented with t( 14;18), as found in our patient. The chromosomal translocation t( 14;18)(q32;q21) represents one of the most common chromosomal aberrations in human B-cell neoplasms and is found on > 80% of follicular lymphomas [37]. In neoplastic cells with this translocation the bcl-2 gene is juxtaposed from 18q21 to transcriptionally active immunoglobulin heavy-chain sequenceson 14q32. Following the translocation, the regulatory influence of the immunoglobulin enhancer segments is thought to lead to an increased level of hybrid bcl-2-Ig RNA [38,39]. The interaction of bcl-2 with other members of the bcl-2 family results in perturbation of the cell cycle and protection of the cell from apoptotic cell death [40-431. Another important feature of the Z-138 cells is the 8;14 translocation. The translocation t(8;14)(q24;q32) is typically found in non-Hodgkin’s lymphoma, particularly in Burkitt’s lymphoma [44,45]. In this translocation, the c-myc oncogene, located on 8q24, is brought under the control of the J, gene at 14q32 and becomes

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1

2

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9

3

4

5

6

7

8

9

10

10

11

11

12

PRADI

pPI-6

PRADl

pDy-12

12

Fig. 7. Southern blot of DNA was hybridized with a PRADl pPI-8 probe (upper panel) and a PRADl pDyl2 probe (lower panel). Upper panel: DNA was extracted from blood derived from patient D-Z-138 (the patient from whom the Z-138 cell line was derived) (lanes 1,4, 7 and lo), Z-138 cells (lanes 2, 5, 8 and 11) and from K562 cells (a CML cell line used as a control) (lanes 3, 5, 8 and 12). Restriction enzymes used were BarnHI (lanes l-3), Bell (lanes 4-6), EcoRI (lanes 7-9) and XbaI (lanes 10-12). Lower panel: DNA was extracted from blood derived from patient D-Z-138 (the patient from whom Z-138 cell line was derived) (lanes 1, 5 and 9) Z-138 cells (lanes 2, 6 and lo), blood leukocytes from a CML patient used as a control (lanes 3, 7 and 11) and from K562 cells (a CML cell line used as a control) (lanes 4, 8 and 12). Restriction enzymes used included EamHI (lanes l-4), EcoRI (lanes 5-8) and Hind111 (lanes 9-12).

activated [46-481. In our case, molecular analysis of the patient’s BM cells did not detect c-myc rearrangement. Nevertheless, activation of this oncogene probably contributed to the rapid cellular proliferation and to the aggressive nature of the disease. Table 2 Cytokine levels in Z-138 cells Cytokine IL-lb IL-6 G-CSF GM-CSF TNF-U TGF-P

Concentration Cell lysates (pg/2 x IO7 cells) Supematant (pg ml-‘) Cell lysates (pg/2 x lo7 cells) Supematant (pg ml-‘) Cell lysates (pg/2 x 10’ cells) Supematant (pg ml-‘) Cell lysates (pg/2 x 10’ cells) Supematant (pg ml-‘) Cell lysates (pg/2 x 10’ cells) Supernatant (pg ml-i) Cell lysates (pg/2 x lo7 cells) Supernatant (pg ml-‘)

0 0 0 0 6758 ND 29 0 0 0 ND 0

Representative data from at least two experiments showing similar results are depicted. In the cases where the cytokine measured values are above or below the sensitivity of the assay, the actual cytokine levels are presented in brackets. ND, not done.

An additional abnormality that could account for the rapidly progressive disease is the 11;14 translocation. The translocation t( 11;14)(ql3;q32) has been observed in a wide spectrum of B-lymphoproliferative diseases and involves the putative oncogene PRADl(bcZ-l/cyclin Dl) that maps to llq13 [49-521. This chromosomal translocation joins the J, locus on 14q32 to the &Z-l locus on 1lq13. Rearrangement or overexpression of bcl-1 is frequently found in lymphoproliferative disorders, particularly in centrocytic/mantle cell lymphoma and it is believed to play a crucial role in the development of this neoplasm [52-541. We found that Z-138 cells express high levels of the 1.7-kb PRADl transcript. The presence of a 9q34 anomaly in Z-138 cells suggested that although Ph’ was not found these cells may express the bcr-abl hybrid gene, which may account of the dismal prognosis for our patient [29]. While this gene is typically found in cells with t(9;22)(q34;qll), cells with the cytogenetic anomaly 9q34, but without a detectable chromosome 22 abnormality, can express bcr-abl. We therefore analyzed Z- 138 cells for the presence of bcr-abl and found no evidence of either bcr-abl messageor of p210bcr-abior p190bcr-ab6.

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Because myeloid hematopoietic growth factors have been found to be produced by cells derived from nonmyeloid lineages and to proliferate in response to these cells [29,30,55-581, we measured the production of such cytokines by Z-138 cells. We found that Z-138 cells do not produce IL-la, IL-6 TNF-a, or TGF-/?. However, they do produce GM-CSF and large quantities of GCSF. Autocrine growth control, whereby a neoplastic cell elaborates a cytokine that supports its own growth, has been observed in several hematologic malignancies [24,31,32,58-611 and is probably an important event in the neoplastic clonal evolution. It is possible that the production of and/or response to one or more cytokines in an autocrine or paracrine fashion further stimulated the neoplastic cells and contributed to the aggressive course of the disease in our patient. We therefore tested the effect of GM-CSF and G-CSF on the proliferation of Z-138 cells. We found that neither cytokine stimulates Z-138 cell growth. These results, in particular the one obtained with G-CSF, are not surprising. Although G-CSF receptors were found on neoplastic B-cell progenitors [62,63] most of the receptor-positive cells do not respond to G-CSF [63]. In conclusion, we describe unique mature B-cell ALL cell line that was derived from a patient whose CLL evolved into an aggressive form of mature B-cell ALL. Z-138 is unusual in that it possessedcytogenetic abnormalities associated with low grade (t(14;18), intermediate grade (t(l1;14)) and high grade (t(8;14)) lymphoproliferative neoplasms. Acknowledgements This study was supported in part by a grant from the National Cancer Institute (PO1 CA 55164). We thank Rod Morgan (GENETRIX, Director, AA Sandberg) for his help in performing the cytogenetic analysis.

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